HIV-1 R5 viruses vary considerably in phenotypes, including macrophage-tropism and sensitivity to neutralizing antibodies. Thus, highly mac-tropic envelopes (envs) exploit low amounts of CD4 and/or CCR5 for infection and contrast with non-mac-tropic R5 envs that require high CD4 levels and infect macrophages inefficiently. The capacity of highly mac-tropic envs to use low CD4/CCR5 suggests that such variants may confer a broader tropism among other CD4+ cell types that express low receptor levels and may have an advantage during virus transmission. Our recent data, using neutralizing monoclonal antibodies and entry inhibitors, strongly suggests that highly mac-tropic R5 envs carry a more exposed CD4 binding site (CD4bs) and may be vulnerable to neutralizing antibodies (nabs) induced by vaccines. We propose to extensively investigate tropism and neutralization sensitivity of diverse R5 envs in the following four aims: Aim 1. The effect of HIV-1 R5 macrophage-tropism on infection of different CD4+ cell populations. We will examine whether highly mac-tropic R5 envelopes confer a broader tropism among CD4+ T-cell populations. Aim 2. Further analysis of the envelope determinants for macrophage infection and use of low CD4 in clade B and non-clade B envelopes. We will analyze clade B and non-clade B envs to identify determinants of tropism and to further examine the role of the CD4 binding loop. Aim 3. Investigation of the impact of R5 env variation in macrophage-tropism on sensitivity to neutralizing antibodies. We will investigate how R5 env variation impacts on sensitivity to HIV-1+ human sera, neutralizing mabs and vaccine induced neutralizing antibodies. Aim 4. Development and characterization of bacterially-produced, unglycosylated protein constructs that mimic the CD4bs. We have designed and produced CD4bs mimics that carry the critical elements of the CD4bs in an unglycosylated peptide that can be produced in bacteria. These CD4bs mimics bind CD4 and b12. Their structure and capacity to induce neutralizing antibodies will be investigated. We expect to provide important insights into (1) the variation of R5 envs for tropism and neutralization sensitivity, (2) the env amino acids and structural determinants involved in varying tropism and neutralization sensitivity and (3) the impact of this variation in the design of novel env-based vaccines. RELEVANCE (See instructions): Our application proposes an extensive investigation into how HIV-1 viruses that transmit vary in their biological properties. Our data will guide us in the development of novel vaccines designed to elicit protection against global HIV-1.